This invention relates to the field of control of cytoskeletal structure and changes in the cytoskeletal structure, especially as it relates to the regulation of cell motility, transformation and tumorigenesis. Specifically the invention relates to human actin-binding regulatory proteins, nucleic acids encoding these proteins, epitopes of these proteins and antibodies specific for these epitopes. The invention also relates to screening methods for the identification of potential drug candidate molecules and the use of such molecules in cancer therapy and the treatment of other disorders of cell motility, cell proliferation, wound healing, growth and division.
The conversion of epithelial cells from sessile, non-dividing cells in monolayers to motile, proliferating cells of invasive carcinomas must be tightly coupled to highly regulated rearrangements of the actin cytoskeleton. In malignant carcinoma tumors, invasion of transformed epithelial cells into the underlying connective tissue occurs by cell migration (References 1-3). Metastasis of carcinoma tumors also involves cell migration from the primary tumor site into blood vessels by diapedesis through the vessel endothelium (Ref. 2).
Migration of metastatic tumor cells was clearly described by Waldeyer in 1872 as amoeboid movement (Ref. 4), a form of cell motility that requires coordinated mobilization and remodeling of the actin cytoskeleton by actin-binding proteins (Refs. 5-10). An initial step in cortical actin cytoskeleton rearrangement includes site-specific actin polymerization onto actin filament ends that have been generated by severing or uncapping of existing filaments (Ref. 11). Two families of actin filament fragmenting/capping proteins are presently recognized, the severin/fragmin/gelsolin family containing shared 125 amino acid repeat domains (Refs. 12-16), and the actin depolymerization factor family of ADF (Ref. 17), depactin (Ref. 18), destrin (Ref. 19), and actophorin (Ref. 20). Severin from Dictyostelium amoebae (Refs. 21,22) and fragmin in Physarum slime molds (Ref. 23) are the earliest phylogenetic examples of actin filament fragmenting proteins. The parallel actin severing protein in mammalian cells is gelsolin, an 80 kDa protein derived from duplication of the ancestral severin gene (Ref 24). A cytoplasmic gelsolin is expressed in epithelial cells, fibroblasts and leucocytes, and secreted plasma gelsolin is present in blood (Refs. 5,6,25). In gelsolin, it is the conservation of severin amino acid sequences that accounts for the actin filament severing activity (Refs. 13,26,27).
Gelsolin is implicated in mammalian cell motility by the demonstration that increased expression of gelsolin in fibroblasts by gene transfection proportionally enhances the rate of migration (Ref. 28). Actin binding protein, ABP 120, has also been implicated in cell motility by functional phenotype analysis (Refs. 29,30). Paradoxically, despite the heightened migratory behavior of invasive tumor cells, gelsolin is extensively down-regulated during transformation of mammary epithelium and fibroblasts (Refs. 31,32).
The following patents and scientific publications may be useful in practicing the full scope of the invention. These patents are incorporated herein by reference in their entirety. The scientific literature is cited to give an indication of the available art known to the skilled artisan in the field. These patents and publications are provided for illustrative purposes.
U.S. Pat. No. 5,374,544 is entitled xe2x80x9cMutated skeletal actin promoter.xe2x80x9dU.S. Pat. No. 5,464,817 is entitled xe2x80x9cMethods for reducing the viscosity of pathological mucoid airway contents in the respiratory tract comprising addministering actin-binding compounds with or without DNAse I.xe2x80x9d
U.S. Pat. No. 5,508,265 entitled xe2x80x9cTherapeutic uses of actin-binding compoundsxe2x80x9d discloses the use of actin-binding compounds, including gelsolin and biologically active fragments thereof in the treatment of actin-related disorders.
U.S. Pat. No. 5,593,964 is entitled xe2x80x9cMethods of treating septic shock by preventing actin polymerization.xe2x80x9d
U.S. Pat. No. 5,656,589 is entitled xe2x80x9cMethod for the reduction of viscous purulent airway contents in the respiratory tract comprising administering actin-binding compounds with or without DNAse I.xe2x80x9d
U.S. Pat. No. 5,851,786 is entitled xe2x80x9cProduct and process to regulate actin polymerization.xe2x80x9d
U.S. Pat. No. 5,071,773 entitled xe2x80x9cHormone receptor-related bioassaysxe2x80x9d discloses assay methods using transcriptional reporter genes generally useful for high throughput screening. Such screens may be adapted for use of assays employing genes encoding actin-binding and regulatory proteins in addition to the steroid hormone receptors which act as transcription factors.
U.S. Pat. No. 5,401,629 discloses further screening methods using readouts based on detecting changes in the transcription of reporter genes engineered to express a detectable signal in response to activation by intracellular signaling pathways.
U.S. Pat. No. 5,482,835 entitled xe2x80x9cMethods of Testing in Yeast Cells for Agonists and Antagonists of Mammal G protein-Coupled Receptorsxe2x80x9d discloses methods for screening;
U.S. Pat. No. 5,747,267 also discloses yeast screens and is entitled xe2x80x9cMethod for Identifying a G Protein-Coupled Glutamate Receptor Agonist and Antagonistxe2x80x9d;
U.S. Pat. No. 5,750,353 entitled xe2x80x9cAssay for Non-peptide Agonists to Peptide Hormone Receptorsxe2x80x9d discloses further screening methods; as does U.S. Pat. No. 5,925,529 entitled xe2x80x9cMethod for Discovery of Peptide Agonistsxe2x80x9d;
U.S. Pat. No. 5,744,303 is entitled xe2x80x9cFunctional Assays for Transcriptional Regulator genesxe2x80x9d; and U.S. Pat. No. 5,569,588 discloses xe2x80x9cMethods for Drug Screeningxe2x80x9d.
Andre, E. A., M. Brink, G. Gerisch, G. Isenberg, A. Noegel, M. Schleicher, J. E. Segall, and E. Wallraff. 1989. J. Cell Biol. 108: 985-995. Is entitled: xe2x80x9cA Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxisxe2x80x9d.
Yin, H. L. et al. 1990. FEBS LETT. 264(1): 78-80 is entitled xe2x80x9cSeverin is a gelsolin phenotypexe2x80x9d.
Jones, J. G., J. Segal and J. Condeelis. 1991. Experientia-Suppl. 59: 1-16 is entitled xe2x80x9cMolecular analysis of aioeboid chemotaxis: parallel observations in amoeboid phagacytes and metastatic tumor cells.xe2x80x9d
Eichinger et al. 1991. J. Cell. Biol. 112(4): 665-76 is entitled xe2x80x9cDomain structure in actin-binding proteins: expression and functional characterization of truncated severin.xe2x80x9d
Prendergast, G. C. and E. B. Ziff 1991. EMBO J. 10(4): 757-66 is entitled xe2x80x9cMbh1: a novel gelsolin/severin-related protein which binds actin in vitro and exibits nuclear localization in vivo.xe2x80x9d
Finidori et al. 1992. J. Cell. Biol. 116(5): 1145-55 is entitled xe2x80x9cIn vivo analysis of functional domains from villin and gelsolin.xe2x80x9d
Eichinger, L. and M. Schleicher. 1992. Biochemistry 31(20) 4779-87 is entitled xe2x80x9cCharacterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.xe2x80x9d
Schnuchel et al. 1995. J. Mol. Biol. 247(1): 21-7 is entitled xe2x80x9cStructure of severin domain 2 in solution.xe2x80x9d
Folger, P. A. 1996. Ph.D. thesis, Cornell University, entitled xe2x80x9cIdentification, isolation and expression of M-severin, a novel actin filament severing preotein in murine carcinoma tumors.xe2x80x9d
Markus et al. 1997. Protein Sci. 6(6): 1197-1209 is entitled xe2x80x9cRefined structure of villin 14T and a detailed comparison with other actin-severing domains.xe2x80x9d
Eichinger, L. et al. 1998. J. Biol. Chem. 273(21): 12952-9 is entitled xe2x80x9cCharacterization and cloning of a Dictyostelium Ste20-like protein kinase that phosphorylates the actin-binding protein severin.xe2x80x9d
Weber, I., Niewohner, J., and Faix, J. 1999. Biochem. Soc. Symp. 65:245-65 is entitled xe2x80x9cCytoskeletal protein mutations and cell motility in Dictyostelium.xe2x80x9d
Despite a longfelt need to isolate the human actin regulatory proteins corresponding to M-severin and M-30 these these proteins have not been provided until the disclosure of the present invention.
Nowhere in these references is there any disclosure, suggestion or even hint of the use of actin-binding and regulatory proteins including human severin (also herein interchangeably referred to as human M-severin) and human M-30, the activities of which are diagnostic for the particular stage of a proliferative disorder, as screens for modulators of the activity of M-severin, M-30 and other members of the actin-binding regulatory molecules, and the use of such modulators as novel drug candidate molecules.
The invention provides isolated native or recombinant human actin-binding regulatory proteins which are expressed in motile, proliferating and invasive cells, and in cells at the site of a wound. Cloning and expression of these proteins in recombinant hosts and methods of purification of the recombinant proteins are provided. Methods are also provided for isolation of each of these proteins from human cells, the raising of antibodies thereto and methods of use of these proteins in actin-binding and actin-severing assays.
In a particular embodiment the protein is human M-severin. In a second embodiment the protein is human M-30. The terms human M-severin and human M-30 are used throughout this specification to designate actin-binding regulatory proteins expressed in motile, proliferating and invasive cells, and in cells at a wound site, and fragments of each which retain actin-binding, actin-severing or regulatory function or any combination of these properties. Human M-severin is a protein which cross reacts with antibodies directed against both Dictyostelium and mouse M-severin and has an apparent MW of aprox. 40,000 as assessed by SDS-polyacrylamide gel electrophoresis. Human M-30 protein is an actin-bundling protein which cross reacts with both anti-Dictyostelium M-30 antibodies and anti-mouse M-30 antibodies and exhibits an apparent MW of approximately 34,000 to 35,000 as assessed on SDS-polyacrylamide gels.
Such native or recombinant proteins and active fragments may be at least 90% homologous to the human M-severin or human M-30. Most preferred for practicing the invention are the native or recombinant proteins and active fragments that are preferably at least 95% to 97% homologous to the human M-severin or human M-30. The optimum native or recombinant proteins and active fragments of the latter class are 97% -, 98% or 99% -100% homologous to the human M-severin or human M-30 proteins.
In yet another embodiment the invention provides a native or recombinant human actin-binding regulatory protein expressed in motile, proliferating and invasive cells, and in cells at a wound site or at the site of a healing wound, which is capable of severing F-actin filaments.
In a further embodiment the invention provides a nucleic acid molecule, including both DNA and RNA molecules, encoding a native or recombinant human actin-binding regulatory protein expressed in motile, proliferating and invasive cells, and in cells at the site of a wound or a healing wound. The nucleic acid may comprise a vector in addition to the sequence encoding the native or recombinant human actin-binding regulatory protein. Such nucleic acids of the present invention encompass natural variants, allelles and polymorphs of the human M-severin and human M-30 genes as well as recombinant molecules encoding these variants, allelles and polymorphic forms.
A polyclonal or monoclonal antibody which specifically binds an epitope of a native or recombinant human actin-binding regulatory protein is also provided. Among these monoclonal and polyclonal antibodies are those which specifically bind human M-severin and human M-30. Single chain antibodies which specifically bind an epitope of human M-severin are also provided.
The invention further provides a method of determining the proliferative status or stage of carcinogenesis of a cell, comprising: providing a cell sample from a cell culture, primary cell isolate or biopsy, assessing the levels of M-severin or M-30, and thereby determining the proliferative status of the cell. The level of M-severin or M-30 may be assessed by a northern blot or western blot techniques, or by cytoimmunohistochemistry.
In yet a further embodiment the invention provides a method of identifying a compound as a modulator of M-severin expression or activity, comprising: providing a cell expressing M-severin protein, contacting the cell with a test compound, assessing the activity of the M-severin in the cell, assessing the activity of the M-severin in an identical cell which has not been contacted with the test compound, comparing the two M-severin activities, and thereby determining whether the test compound is a modulator of M-severin activity.
In still yet a further embodiment the invention provides a method of identifying a compound as a modulator of M-30 expression or activity, comprising: providing a cell expressing M-30 protein, contacting the cell with a test compound, assessing the activity of the M-30 in the cell, assessing the activity of the M-30 in an identical cell which has not been contacted with the test compound, comparing the two M-30 activities, and thereby determining whether the test compound is a modulator of M-30 activity.
In yet another embodiment the invention provides a method of treating a mammalian cell in a stage of carcinogenensis comprising: administering an effective amount of a compound which modulates M-severin expression or activity such that carcinogenesis is modulated. Among these compounds are molecules that modulate the actin-binding or actin-severing activity of the human M-severin. Other compounds of this embodiment may function by modulating the transcription of the M-severin.
Further in yet another embodiment the invention provides a method of treating a mammalian cell in a stage of carcinogenensis comprising: administering an effective amount of a compound which modulates M-30 expression or activity such that carcinogenesis is modulated. Among these compounds are molecules that modulate the actin-binding or actin-severing activity of the human M-30. Other compounds of this embodiment may function by modulating the transcription of the M-30 .
It should be understood that throughout the specification the terms M-severin and M-30 refer to mammalian severin and M-30, of which human severin and human M-30 are species. Further, the terms M-severin and M-30 refer to both native and recombinant forms of the proteins whether isolated from human cells or from recombinant hosts, which may be eukaryotic or prokaryotic hosts carrying the recombinant nucleic acid sequence encoding M-severin or M-30 .